PROJECT SUMMARY Despite advancements in clinical treatment and screening, breast cancer remains the second most deadly and most frequent cause of cancer in women. BRCA1 related cancer is associated with dramatically increased risk of cancer occurrence. Most BRCA1 related breast cancers will be ?triple-negative?, with no targeted therapies, poor prognosis, and decreased overall survival. Pre-neoplastic changes in BRCA1 mutation carrier epithelium have been well studied, as well has the tumor microenvironment. However, BRCA1 germline mutations are likely to affect all cells in the breast, not just the epithelium. Less well studied, and the focus of this proposal, are the pre-neoplastic changes that occur in the stromal cells of the breast microenvironment, and their contributions to cancer initiation. The overarching hypothesis of this proposal is that the breast microenvironment is distorted by BRCA1 mutations and contribute to cancer initiation. A persistent challenge in assaying the stroma of the breast microenvironment is the diversity and heterogeneity of this tissue compartment. To overcome this challenge, our lab has performed single cell RNA sequencing (scRNAseq) of 81,023 stromal and epithelial cells from a cohort of high-risk BRCA1 mutation carrier and control patient surgical breast samples. scRNAseq has the resolution to identify specific cell populations in the breast microenvironment and how they change in the preneoplastic microenvironment. Using this highly multidimensional data, this proposal aims to 1) describe altered paracrine interactions in the pre-neoplastic breast and to 2) identify how microenvironment composition may be distorted in BRCA1 mutation carrier breasts. Using scRNAseq and in vitro organoid models, preliminary work has defined a pro-proliferative paracrine interaction of pericytes with basal epithelium. Additionally, this study uses scRNAseq data to develop a novel flow cytometry strategy to specifically isolate pericytes to demonstrate that pericytes have multipotent mesenchymal potential. This study proposes that these multipotent pericytes may serve as the stromal mesenchymal stem cell progenitor. To assess how these interactions contribute to breast cancer initiation, this study proposes two in vivo humanized mouse models that allow growth of oncogenic patient breast epithelium with defined patient-derived stromal populations. If successful, this study will provide insights into paracrine interactions that have been previously undefined but may have roles in breast cancer initiation. Furthermore, identification of a stromal progenitor may yield new biomarkers and targets for high-risk breast cancer.